The long-term goal is to understand the structure-function mechanisms that control actin cytoskeleton dynamics during cellular processes such as cell motility, endocytosis and intracellular trafficking. The emphasis during this period will be on actin filament nucleation. In cells, actin exists in regulated equilibrium between its monomeric (G-actin) and filamentous (F-actin) forms, with F-actin accounting for most cellular functions of actin. Nucleation is rate limiting during actin filament assemble. So, eukaryotic cells (and certain pathogens) use filament nucleators to stabilize actin polymerization nuclei (dimers, trimers and tetramers). Filament nucleators control not only the time and location for actin polymerization, but also the specific type of actin networks that they generate. Known filament nucleators include the Arp2/3 complex, formins, Spire, Cobl, VopL/VopF and Lmod. These molecules are generally unrelated, yet with the exception of formins they all use the WASP- Homology Domain 2 (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. Building upon a deep understanding of the W-actin interaction acquired during the previous period of this grant, the current proposal aims to understand the structure-function mechanisms of W-dependent actin filament nucleation. The specific aims are: 1. To understand the mechanism of actin filament nucleation by NPFs-Arp2/3 complex. The Arp2/3 complex, together with its large family of W-based Nucleation Promoting Factors (NPFs), is unique among filament nucleators in that it generates branched actin networks. The crystal structure of the inactive complex is known. Here, the focus will be on the activated complex, a challenging structure whose study will require the design and implementation of innovative strategies. 2. To understand the nucleation mechanism of VopL, a powerful nucleator and virulence factor produced by the gastroenteritis causing pathogen Vibrio parahaemolyticus. The study of VopL has general relevance to other filament nucleators, such as Cobl and Spire, which like VopL are based on tandem repeats of W domains. 3. To understand the nucleation mechanism and cellular function of Lmod, a muscle-specific actin filament nucleator discovered during the previous period of this grant. Lmod, which is unrelated to other filament nucleators, contains three actin-binding sites, one of which is a W domain. Extensive preliminary results lay the groundwork for these studies. Collaborative studies are also underway to understand the cellular function of Lmod, and to determine the structure of activated Arp2/3 complex using single-particle electron microscopy. This research will expand our understanding of the molecular bases of actin filament nucleation in health and disease. PUBLIC HEALTH RELEVANCE: The actin cytoskeleton is involved in most cellular function, including endo/exocytosis, cell motility, and the maintenance of cell shape and polarity. The filament nucleators studied here play critical roles in health and disease. Thus, Arp2/3 complex-mediated nucleation is a crucial factor for the migration of metastatic cancer cells, VopL is a virulence factor of the gastroenteritis causing pathogen Vibrio parahaemolyticus, and Lmod is involved in myofibril morphogenesis in heart muscle. The knowledge gained here will be of great relevance to understanding the molecular mechanisms of disease.